The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

Unfolding of mRNA secondary structure by the bacterial translation initiation complex

Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.     

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.     

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.     

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.     

Inability of Agrobacterium tumefaciens ribosomes to translate in vivo mRNAs containing non-Shine-Dalgarno translational initiators

Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli. Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo. A phage T7 gene 10 sequence, called "epsilon" (epsilon), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells. In this study the epsilon, together with three other nucleotide sequences derived from the 5' non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A. tumefaciens cells. The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA. To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E. coli) was co-expressed in A. tumefaciens together with the cat gene placed under the translational control of the above sequences. Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A. tumefaciens cells. The inability of A. tumefaciens ribosomes to translate mRNAs devoid of SD sequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E. coli and A. tumefaciens.     

Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.     

Coding theory based models for protein translation initiation in prokaryotic organisms

Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism. The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook). We tested the E. coli based coding models on 5' untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E. coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50. The model identified regions on the 5' untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most. These regions correspond to the Shine-Dalgarno domain and the non-random domain. Applying the EC coding-based models to B. subtilis, and S. aureus Mu50 yielded results similar to those for E. coli K-12. Contrary to our expectations, the behavior of S. typhimurium LT2, the more taxonomically related to E. coli, resembled that of the non-translated sequence group.     

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.